Background <p>Radiotherapy (RT) is an essential treatment for colorectal cancer (CRC), yet the factors influencing radiosensitivity remain unclear. In the quest to enhance the therapeutic efficacy in CRC, the interplay between genetic mutations and RT sensitivity has emerged as a pivotal yet enigmatic area.</p> Methods <p>We harness the fidelity of patient-derived organoids (PDOs) to dissect the molecular landscape of radiosensitivity, with a particular emphasis on BRAF^V600E mutations. To further investigate, a cohort of 9 BRAF^V600E-mutant and 10 BRAF wild-type PDOs is constructed to systematically assess the radiobiological traits of BRAF^V600E-mutant CRC, including morphology, cell viability, and DNA damage, while also evaluating their responses to chemotherapy and chemoradiotherapy.</p> Results <p>Our systematic investigation unveils a profound correlation between BRAF^V600E mutation status and radioresistance, which is validated by clinical treatment responses. Intriguingly, BRAF^V600E-mutant PDOs exhibit reduced sensitivity to conventional chemotherapy, yet demonstrate an enhanced response to combined chemoradiotherapy, characterized by increased apoptosis. The results are validated through in vivo analyses using patient-derived organoid xenograft mouse models and aligned with patient clinical outcomes.</p> Conclusions <p>This study outlines the distinct radiobiological profile of BRAF^V600E-mutant CRC, underscoring the critical role of radiotherapy in comprehensive treatment strategies. This work not only advances our molecular understanding of CRC but also paves the way for precision medicine, offering valuable insights for therapeutic decision-making in the clinical management of BRAF^V600E-mutant CRC.</p>